A generalizable method for the purification and biophysical characterization of bacterial membrane receptors.

Authors

Federico Bosetto, Maria Zacharopoulou, Mark Bycroft, Monika Kish, Giovanna Zinzalla, Pamela J E Rowling, Stephen H McLaughlin, Jonathan J Phillips, Laura S Itzhaki, Ioanna Mela

Published in

Protein science : a publication of the Protein Society. Volume 35. Issue 11. Pages e70825.

Abstract

Membrane-embedded bacterial receptors are challenging to express and purify in soluble form, yet their isolated domains are essential tools for structural and ligand-discovery studies. Pseudomonas aeruginosa relies on the TonB-dependent heme receptor HasR for iron acquisition, a process central to its pathogenicity. Here, we report a robust strategy for the recombinant expression, purification, and biophysical characterization of the two soluble HasR domains directly involved in heme uptake: the N-terminal plug and the Secretin/TonB short N-terminal domain. Each domain was expressed individually in Escherichia coli and purified to homogeneity, adopting well-folded conformations as confirmed by circular dichroism, NMR spectroscopy, and mass spectrometry. We then engineered a fusion construct containing both domains and systematically evaluated multiple solubilization tags. A GST-His dual-affinity strategy enabled efficient purification of the construct, whereas His6-tag alone resulted in insoluble protein and HLT-tag fusions suffered from non-specific proteolysis. Biophysical analyses revealed that the Secretin/TonB short N-terminal domain remains stably folded within the fusion construct, while the N-terminal plug domain becomes partially disordered, a finding further supported by hydrogen/deuterium exchange mass spectrometry. Together, these results establish a generalizable workflow for producing soluble receptor domains from membrane proteins and provide validated HasR constructs suitable for downstream ligand-screening applications, including aptamer and nanobody discovery.

PMID:
42876396
Bibliographic data and abstract were imported from PubMed on 15 Oct 2026.

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